Current antiretroviral drugs keep HIV replication in check, allowing infected individuals to survive for decades. However, antiretroviral therapy does not clear the virus from the body, requiring life-long drug administration to prevent relapse. Chronic antiretroviral drug exposure contributes to clinical metabolic disturbances, organ damage and drives drug resistance, underscoring the need for new pharmaceutical agents to combat HIV disease. To address these critical issues, we have launched a drug discovery campaign targeting the HIV-1 virulence factor, Nef. Unique to primate lentiviruses, Nef is critical for high-titer HIV-1 replication in vivo, immune escape of HIV-infected cells, and AIDS progression. Using a high- throughput screening assay developed during the previous grant period, we have identified several classes of Nef inhibitors with potent antiretroviral activity against Nef-dependent HIV replication in primary human macrophages. In this renewal application, we propose to explore the molecular mechanism of action of these compounds, and to test their efficacy in vivo with the following Specific Aims: Aim 1. Leverage new Nef-binding compounds to map hot spots for antagonist binding. We have identified seven discrete chemical scaffolds with nanomolar to single-digit micromolar activity against Nef-dependent enhancement of HIV replication in vitro. In this Aim, we propose to combine X-ray crystallography and site-directed mutagenesis to map their binding sites on Nef. We predict that these diverse scaffolds will bind to a common 'hot spot' for Nef inhibitor action, which will enable structure-based analog design for in vivo testing. Aim 2. Test the hypothesis that Nef antagonists function by preventing Nef dimerization and interactions with host cell effector proteins. Nef interacts with a diverse group of host cell proteins involved in cell-surface receptor trafficking (e.g., AP-1 and AP-2) and signal transduction (e.g., cytoplasmic protein kinases). Using a cell-based fluorescence complementation assay, we will probe inhibitor impact on Nef homodimerization as well as interactions with a diverse panel of effector proteins, and correlate these effects with Nef functions in HIV-infected cells. These studies will identify the chemical scaffolds that affect as many Nef interactions and functions as possible. Aim 3. Test the hypothesis that Nef antagonists can suppress HIV replication and T-cell loss in a humanized mouse model of AIDS. Humanized mice (NSG mice reconstituted with human immune cells) infected with Nef-deleted HIV-1 exhibit dramatically lower viral loads and substantially less T cell depletion than mice infected with wild-type virus, validating their use to explore Nef inhibitor action at the whole animal level. We will determine the effect of Nef inhibitors on HIV replication, CD4+ T cell loss and immune system function in this model system. These studies represent the first evaluation of small molecule inhibitors of HIV-1 Nef function in vivo, where they are predicted to suppress Nef-dependent enhancement of HIV replication and CD4+ T cell loss.